ps20 amino acid sequences
Urogenital sinus derived growth inhibitory factor is a protein having growth-inhibitory and antiprotease properties. The present invention relates to amino acid and nucleotide sequences for urogenital sinus derived growth inhibitory factor.
[0001] This application claims the benefit of U.S. Provisional Application No. 60/008,348 filed Dec. 7, 1995. U.S. Provisional Application No. 60/008,348 was converted to U.S. patent application Ser. No. 08/761,248 on Dec. 6, 1996. This application is a divisional and continuation in part of U.S. patent application Ser. No. 08/761,248 (now U.S. Pat. No. ______).
GOVERNMENT INTEREST BACKGROUND OF THE INVENTION[0003] 1. Field of the Invention
[0004] The present invention relates to a urogenital sinus derived growth inhibitory factor, ps20. More specifically, the present invention relates to uses of the factor and to the amino acid and nucleotide sequence of said factor. The present invention also relates to antibodies which bind to said factor.
[0005] 2. Description of the Prior Art
[0006] Epithelial differentiation patterns are induced by stromal cells in most eukaryotic tissues including lung, breast, stomach, skin, eye, and prostate gland. In prostate development, ductal morphogenesis and epithelial acini differentiation result from stromal induction. In heterotypical tissue-tissue recombinants, mesenchyme from fetal urogenital sinus (anlagen of the prostate gland) induces bladder epithelial cells to undergo ductal morphogenesis and differentiation to a prostatic epithelial phenotype, capable of expressing prostate specific proteins.
[0007] Prostate ductal morphogenesis is characterized by a stromal-induced epithelial cell proliferation. Following this, epithelial cytodifferentiation (to the secretory phenotype) is correlated with a cellular quiescence which also requires stromal interaction. In most cells, differentiated gene expression is associated with a reduced cell proliferation. In cell culture, growth stimulatory activities (to epithelial cells) have been observed in the conditioned medium from prostate stromal cells, including bFGF and NGF-like factors.
[0008] Progress has been made in the identification of keratinocyte growth factor (KGF) as a likely candidate stromal-derived factor. KGF expression is restricted to stroma and is androgen regulated in the prostate. KGF mediates, in part, the stromal induction of seminal vesicle epithelium proliferation. TGF-&bgr; and TGF-&bgr; receptors are negatively regulated by androgen in the prostate. TGF-&bgr;s are expressed in development, however, their role in prostate development is not clear. Growth inhibitory activities secreted from prostate stromal cultures have been reported by the inventor's laboratory group and others, not attributed to known inhibitory factors, including the TGF-&bgr;s. This growth inhibitory activity is attributable to the urogenital derived sinus growth inhibitory factor (UGIF) ps20 protein, which also induces protein synthesis and alters phenotypic morphology of target epithelial cells. As with all other growth regulatory proteins, ps20 is not specific to prostate, but is also expressed in mesenchymal and smooth muscle cells in other tissue. The developmental pattern of prostatic ductal morphogenesis followed by epithelial differentiation likely involves the timed expression of a variety of positive and negative growth regulatory factors.
[0009] Studies with rat and human prostatic smooth muscle cell lines show androgen-stimulated proliferation with physiological concentrations (5-10 mM) of androgen. These observations together indicate urogenital sinus mesenchyme and adult smooth muscle cells may express genes fundamental to stromal-epithelial interactions in the prostate gland.
[0010] Progress has been limited in identification of stromal-derived regulatory proteins and their mechanisms due to technical difficulties in the isolation and culture of androgen responsive stromal cell lines, difficulties in the biochemical analysis of secreted or extracellular matrix proteins, and the relative unavailability of tissue-specific stromal cell cDNA libraries.
[0011] Benign prostatic hyperplasia (BPH) and prostate cancer are disorders of prostatic epithelial growth and differentiation. BPH disorders are perhaps most relevant to stromal-epithelial interactions. BPH initiates from localized stromal cell proliferation. The initiation of BPH has been termed a “reawakening” of the inductive potential of the prostate stroma and a spontaneous reversion of the stroma to an embryonic state. Accordingly, the abnormal proliferation of stromal cells in the periurethral region can induce the ingrowth and abnormal formation of acini from adjacent epithelial cells.
[0012] During prostate carcinogenesis, carcinoma progression patterns involve stromal-epithelial interactions. Prostatic carcinoma is typified by progression from an androgen responsive state to an androgen insensitive state which no longer responds to anti-androgen therapy. Some evidence exists to suggest that progression to androgen insensitivity results from altered gene expression in stromal cells. In the Dunning rat prostate carcinoma, the type of stroma can induce the adjacent epithelium to exhibit exon switching of FGF receptors (FGFRc2 IIIb to IIIc) which imparts androgen insensitive proliferation to these epithelial cells. Dunning tumor prostate carcinoma cell proliferation was inhibited by 7-fold when recombined with normal seminal vesicle or urogenital sinus mesenchyme. The recombined carcinoma cells showed an alteration in phenotypic morphology. When recombined with normal mesenchyme, carcinoma cells exhibited a tall, columnar cell shape, typical of a differentiated secretory epithelium as compared to the typical squamous/cuboidal undifferentiated phenotype in wild-type Dunning tumor. In this regard, it is of interest that smooth muscle is absent from Dunning prostatic tumor. In addition, the pattern of carcinoma formation can be influenced by the origin of the associated stromal cells. Recombination of bladder transitional cell carcinoma with normal urogenital sinus mesenchyme resulted in the formation of a glandular adenocarcinoma phenotype typical of prostate. Tissue-tissue recombination studies to produce prostatic tumors in mice requires transformation of mesenchyme (with myc and ras) to produce prostatic adenocarcinoma typical of the human phenotype. Conversely, the inoculation of fibrosarcoma tumorigenic stromal cells with non-tumorigenic normal epithelial cells into nude mice resulted in a mixed carcinoma-fibrosarcoma. Together these studies indicate prostatic carcinoma epithelium is responsive to the stromal environment and that progression and overall phenotype of prostate carcinoma is dependent to some degree on stromal interaction. It follows that key proteins involved in mechanisms of stromal-epithelial interactions will be of significance to the study of prostate proliferation diseases.
[0013] Balance of protease and protease inhibitor function is involved in modeling of tissues, extracellular matrix (ECM) compositions, and growth factor activation processes. Proteases play a significant role in embryogenesis, extracellular matrix modeling/remodeling and in tumorigenesis involving abnormal proliferation, promotion of tumor invasion, and formation of metastasis. It is well-established that metalloproteinases are overexpressed in most neoplastic diseases including breast cancer, colon cancer, neuroblastomas, and prostate cancer. Cysteine proteases, including Cathepsins B and D, are elevated in many cancer metastases, including prostate cancer. Significant to tumor progression, due to their induced cascade of effects are the plasminogen activator (PA) proteases. Plasminogen activators are serine proteases which convert inactive plasminogen to the active form, plasmin. Plasmin in turn exhibits broad proteolytic trypsin-like effects on ECM components including glycoproteins, proteoglycans (including heparin and heparin-sulfates), and gelatins. Plasmin also activates a variety of EMC bound growth factors from latent to active forms including the TGF-&bgr;s. The urokinase-type PA and tissue-type PA have been the most extensively studied PAs. Urokinase PA is primarily involved in tissue modeling-remodeling activities, whereas tissue PA is most active in blood clot lysis.
[0014] Since plasminogen is present in all tissues and fluids, local effects of plasmin are mediated by local expression of PA. Urokinase PA is secreted as an inactive pro-form which binds with high affinity to a membrane-anchored specific receptor where it is cleaved to the active form and remains (on cell surface) for several hours. On the cell surface, urokinase PA has a focal effect resulting in local acceleration of plasmin activation by approximately 40-fold. Plasmin activity is elevated in the focal environment to the cell surface expressing active urokinase. Focal plasmin effects degradation of ECM and activates metalloproteinases (procollagenases, prostromelysin, elastase). Accordingly, secretion of small amounts of urokinase PA results in a focal plasmin cascade to effect a spectrum of other enzymes and factors. Urokinase PA is inhibited by PA inhibitors (PAIs) which are serine protease inhibitors. Local actions of PAs (and other proteases) have been implicated in a wide array of developmental processes through highly regulated mechanisms. PAs, PAIs, and proteases are each regulated by hormones and growth factors.
[0015] In addition to functions in development, urokinase PA is elevated in most tumor metastases. Elevated urokinase PA leads to down stream activation of proteases and growth factors with increased tumor invasion, increased tumor volume, and increased cell proliferation rate. In the prostate, the study of proteases and inhibitors have focused primarily on carcinoma progression. PA activity is higher in prostate carcinoma than in normal tissue and the urokinase PA form is primarily associated with progression. Urokinase PA is elevated in prostate bone metastasis relative to primary tumor site. Urokinase PA is overexpressed in Dunning, Nobel, Lobund-Wistar, and Fisher-334 prostatic tumors. Moreover, urokinase PA is the predominant PA secreted by the PC-3 and DU-145 human prostatic carcinoma cell lines and these cell lines exhibit the urokinase PA cell surface receptor. Studies by the inventor have used the PC-3 cell line to identify and purify ps20 secreted from fetal urogenital sinus mesenchymal cells. Metastasis of PC-3 in nude mice was shown to be blocked by mutated urokinase PA or urokinase PA receptor blocking antibodies.
[0016] Direct evidence shows growth inhibition of cancer cells by urokinase PA inhibitors and other protease inhibitors. A synthetic urokinase PA inhibitor (p-aminobenzamidine) inhibited the progression of DU-145 human prostate carcinoma in SCID mice and cell proliferation in culture in a dose-dependent manner (64% decreased tumor volume). The protease inhibitor actinonin inhibited mammary tumor progression (both non-metastatic and metastatic types) in collagen gels. Batimastat, a matrix metalloproteinase inhibitor, inhibited organ invasion in lung (72% decrease in tumor volume) of two human colon carcinomas. In human prostate, decreased expression of acid cysteine proteinase inhibitor (ACPI)(cathepsin inhibitor) was observed in BPH tissue relative to normal. No expression of ACPI was found in human prostatic adenocarcinoma tissue. Accordingly, balances of proteases and protease inhibitors may affect proliferation in human BPH and carcinoma.
[0017] U.S. Pat. No. 5,196,334, incorporated by reference herein, describes the isolation and partial characterization of urogenital sinus derived growth inhibitory factor, UGIF (ps20). However, the amino acid and nucleotide sequence of ps20 has not heretofore been described. Additionally, antibodies to ps20 have also not heretofore been described. Moreover, the gene sequences from a variety of different sources including human have not heretofor been proven.
SUMMARY OF THE INVENTION[0018] The present invention relates to the amino acid and nucleotide sequences of a urogenital sinus derived growth inhibitory factor. Accordingly, provided herein is an amino acid sequence which codes for urogenital sinus derived growth inhibitory factor, UGIF (ps20). The ps20 of the present invention has protease inhibitory function. Also provided herein is a nucleotide sequence which codes for the urogenital sinus derived growth inhibitory factor protein. Also provided herein are antibodies which bind to urogenital sinus derived growth inhibiting factor, ps20.
[0019] In partcular, the present invention relates to an isolated DNA molecule encoding a urogenital sinus derived growth inhibitory factor. The inventor has provided numerous species of this factor in the examples. The DNA may be any form of DNA, such as cDNA or genomic DNA. It also may be derived from any biological source in which the factor is present, including human DNA, mouse DNA, or rat DNA as shown in the examples. In particular, the DNA may be any of those shown in the Sequence Listing for cDAN or genomic DNA derived from human, mouse or rat biological sources.
[0020] The present invention also relates to an isolated DNA molecule encoding a WAP four-disulfide core domain 1 gene. As discussed herein, this designation identifies the family of genes which encodes urogenital sinus derived growth inhibitory factor (also known as “ps20.”). In particular, the gene may be the human gene WFDC1, or it may be the mouse gene Wfdc1.
[0021] The present invention also relates to the polypeptide family known as recombinant urogenital sinus derived growth inhibitory factor. The term “recombinant” is given its normal meaning to those of skill in the art of gene cloning, and would include any genetically isolated gene or DNA fragment which is isolated independent from the native DNA in which it is found in nature. Such isolation may involve cloning vectors, or it may involve fragmentation of the DNA. Regardless of the manner that the recombinant protein is produced, it may be derived from any biological source in which it is found, and includes the human, mouse and rat proteins.
[0022] With the availability of highly purified recombinant proteins made possible by this invention, the invention discloses antibodies immunologically recognizing urogenital sinus derived growth inhibitory factor. Such antibodies may particularly be polyclonal or monoclonal antibodies.
[0023] Also provided by the present invention are methods of detecting the presence of DNA encoding a urogenital sinus derived growth inhibitory factor in a sample. These methods comprise labeling any of the DNA molecules encoding the factor, or a fragment of the factor. The labeled DNA or fragment is then contacted with a sample containing a target. The method is carried out under conditions known well to those of skill in the art of DNA-DNA hybridization to allow the target DNA to hybridize to the labeled DNA. The method then calls for determining whether or not hybridization has taken place by detecting the presence of the labeled DNA or labeled fragment is hybridized to the target DNA. Labeled DNA or labeled RNA derived from the DNA template may be used to detect messenger RNA (mRNA) through the use of Northern blot analysis, ribonuclease protection assay (RPA) and in situ localization of mRNA in cells using appropriately prepared tissue sections. Moreover, labeled or unlabeled DNA or derived RNA may be used in preparation of a microarray chip used for the detection of the gene or cDNA or mRNA derived from this gene. In some cases the labeling will be done using radioactive markers and in others, fluorescent markers, or combinations of such markers. In a particular embodiment, a kit for detecting the presence of DNA encoding a urogenital sinus derived growth inhibitory factor utilizing such methods will include labeled DNA and hybridization reagents.
[0024] Similarly, the present invention disclsoes methods of detecting the presence of a urogenital sinus derived growth inhibitory factor in a protein sample comprising labeling any of the antibodies of the invention (or immunologically reactive fragments of these antibodies). Then, the labeled antibody or fragment is contacted with a sample containing protein in a manner known by those of skill in the art of immunology to be conducive to antibody-antigen cross reactivity. Finally, the method provides for determining if cross reactivity takes place by detecting the presence of the labeled antibody or fragment. The labeling again may be radiolabeling or fluorescent labeling, among others.
[0025] There is also provided by the present invention a method of detecting the presence or localization of DNA encoding a urogenital sinus derived growth inhibitory factor in a chromosome. The method comprises labeling any of the DNA molecules of the invention (or a fragment of same), such as by radiolabeling, fluorescent labeling or biotin labeling. Then, the labeled DNA or fragment is contacted with a chromosome in a manner known by those of skill in the art to be conducive to DNA-DNA hybridization. Once hybridiazation with the chromosome is accomplished, the presence or localization of the hybridized DNA within the chromosomal DNA encoding the factor is determined.
[0026] The present invention also provides biologically functional vectors comprising DNA molecules encoding a urogenital sinus derived growth inhibitory factor. These vectors may have DNA derived from any suitable biological source including but not limited to human DNA, mouse DNA or rat DNA. These vectors may be incorporated into a host cell, which cell may be either prokaryotic or eukaryotic.
[0027] Such cells having such vectors provide methods of making a recombinant urogenital sinus derived growth inhibitory factor.. These methods comprise culturing such a cell in a medium, harvesting the cell from the culture or harvesting the cell culture medium (dependning upon the greatest source of the recombinant factor. Then, the factor is extracted from the cell or the medium using techniques known well to those of skill in the art of fermenting recombinant proteins.
[0028] Also provided by the invention are methods of treating stromal cells to affect their growth and differentiation characterisitics or phenotype. A stromal cell for purposes of the invention is a fibroblast, myofibroblast, smooth muscle cell, or any precursor cell to such a cell type, or any cell type that differentiates into such a cell type. These methods comprise contacting smooth muscle cells with a urogenital sinus derived growth inhibitory factor. The smooth muscle cells may be any such cells including but not limited to such cells which are vascular cells, tumor cells, and any organ smooth muscle cells (such as those in the prostate gland).
[0029] Also provided are methods of genetic therapy comprising treating a patient with a gene therapy vector capable of transfecting the patient with a gene encoding the factors of the invention. The therapy can be one designed to permanently transfect the patient, or can be designed to only affect a temporary transfection for a time suitable to achieve the desired therapeutic effects. The therapy may one designed to affect any suitable therapy, including but not limited to vascular therapy or cancer therapy.
[0030] These and other advantages of the present invention will become apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS[0031] FIG. 1. Rat ps20 cDNA and the deduced amino acid sequence.
[0032] FIG. 2. Alignment of four disulfide core domain of ps20 with other family members.
[0033] FIG. 3. Western analysis with ps20 peptide antibody.
[0034] FIG. 4. Immunohistochemical localization.
[0035] FIG. 5. Immunolocalization of ps20 in human benign prostatic hyperplasia.
[0036] FIG. 6. Immunolocalization of ps20 in human poorly differentiated carcinoma.
[0037] FIG. 7. Sequence ID No. 1, rat ps20 cDNA nucleotide sequence.
[0038] FIG. 8. Sequence ID No. 2, rat ps20 amino acid sequence.
[0039] FIG. 9. Sequence ID No. 3, human ps20 cDNA nucleotide sequence.
[0040] FIG. 10. Sequence ID No. 4, human ps20 amino acid sequence.
[0041] FIG. 11. Human ps20 cDNA nucleotide sequence and deduced amino acid sequence.
[0042] FIG. 12. A. Localization of human PS20 on chromosome 16q24.2-.3. B. Ideogram showing localization of ps20 relative to banding pattern.
[0043] FIG. 13. A. Genomic organization of mouse PS20 gene. B. Sequence of the mouse PS20 gene.
[0044] FIG. 14. Comparison of human, rat, and mouse ps20 amino acid sequences.
[0045] FIG. 15. ps20 alters morphology of PS-1 adult rat prostate stromal cells in the presence of serum.
[0046] FIG. 16. ps20 promotes spheroid formation in stromal cells: COS and PS-1.
[0047] FIG. 17. ps20 promotes migration of stable transfectant COS cell lines.
[0048] FIG. 18. TGF-&bgr;1 induces expression of smooth muscle marker proteins in PS-1 adult rat prostate stromal cells.
[0049] FIG. 19. ps20 inhibits expression of polymerized smooth muscle &agr;-actin in PS-1 cells.
[0050] FIG. 20. ps20 reduces levels of monomeric SM &agr;-actin in PS-1 cells.
[0051] FIG. 21. ps20 inhibits TGF-&bgr;1 induced expression of smooth muscle markers in PS-1 cells.
[0052] FIG. 22. The inhibition of TGF-&bgr;1 mediated differentiation of PS-1 cells by ps20 is dose-dependent and species-independent.
[0053] FIG. 23. ps20 mRNA expression in adult rat prostate smooth muscle cells is stimulated by TGF-&bgr;1.
DETAILED DESCRIPTION OF THE INVENTION[0054] The isolation and characterization of ps20 is disclosed in U.S. Pat. No. 5,196,334, and U.S. Pat. No. 5,496,800, incorporated by reference herein. The present invention provides nucleotide and amino acid sequences of ps20. As used herein, the term “nucleotide sequence” includes polynucleotides and/or oligonucleotides and refers to a plurality of joined nucleotide units formed from naturally-occurring bases and cyclofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits. “Nucleotide sequence” also refers to purine and pyrimidine groups and moieties which function similarly but which have non naturally-occurring portions. Thus, nucleotide sequences may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species. They may also contain altered base units or other modifications, provided that biological activity is retained. Nucleotide sequences may also include species which include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the cyclofuranose portions of the nucleotide subunits may also occur as long as biological function is not eliminated by such modifications.
[0055] As will become apparent, cloning and sequencing of ps20 reveals that the protein has protease inhibitory function. Therefore, ps20 and its nucleotide sequence may be used in a number of applications wherein protease inhibition is desirable. These include, but are not limited to inhibition or regulation of cell proliferation, inhibition or regulation of cancer cell proliferation, inhibition or regulation of cancer metastasis, regulation of biological activities of other growth factors that are activated by protease action, regulation of extracellular matrix proteins resulting in alteration in cell proliferation and/or cell differentiation (altered gene expression) and/or cell morphologies or any other cell function regulated by extracellular matrix, activation or inactivation of growth factor activities associated with protease action. Diseases or conditions responsive to ps20 include, but are not limited to prostate cancer and metastasis, breast cancer and metastasis, ovarian cancer and metastasis, transitional cell carcinoma and metastasis, renal cell carcinoma and metastasis, bronchogenic carcinoma (lung cancer) and metastasis, colorectal carcinoma and metastasis, endometrial (uterine) carcinoma and metastasis, malignant melanoma and metastasis hepatocellular carcinoma (kidney cancer) and metastasis, pancreatic cancer and metastasis, testicular seminomas and nonseminoma germ cell tumors and metastasis, cervical cancer and metastasis, esophageal squamous cell carcinoma and metastasis, gastric carcinoma and metastasis, atherosclerosis, restenosis after angioplasty, vascular smooth muscle proliferation associated with vascular wall injury, benign prostatic carcinoma, wound healing, and chronic inflammation. Those skilled in the art will be able to ascertain suitable doses of ps20 to achieve the desired protease inhibition function using known pharmacokinetic techniques.
[0056] Additionally, the nucleotide sequence that codes for ps20 may be used as a diagnostic tool for assessing risk of developing prostatic diseases, such as, but not limited to, prostate cancer and benign prostatic hyperplasia. The DNA sequence coding for the ps20 may be isolated and compared to the sequence found in normal and/or at-risk individuals. In the diagnostic assay, “at risk” individuals are those who have or may develop prostatic disease. Additionally ps20 and its nucleotide sequence can be used to prevent a number of diseases or disorders. Those skilled in the art will be able to determine appropriate preventative doses. Also, the ps20 nucleotide sequence may also be used to screen for cardiovascular diseases such as, but not limited to, arteriosclerosis and restenosis. The nucleotide sequence of the present invention may be used to construct recombinant proteins having the amino acid sequence of rat, mouse or human ps20, for example. As used herein, the protein UGIF is referred to also as “ps20.” The gene encoding that protein is named (HUGO Nonenclature Committee, University College, LONDON, UK). WAP four-disulfide core domain 1.” It is given the symbol “WFDC1” in humans and “Wfdc1” in mice. Additionally, proteins having minor modifications may also be constructed using the nucleotide sequence of the present invention. Also provided are vectors comprising the nucleotide sequence of the present invention.
[0057] Also made possible by the present invention are methods of treating cardio vascular disease and other diseases and disorders where there are alterations in smooth muscle biology or phenotype from that of normal, undiseased tissue.
[0058] Also provided herein are antibodies to ps20. The antibodies can be used to identify and enumerate ps20-bearing cells, or can be used to isolate or quantitate the amount of ps20 in body fluids. For this purpose, antibodies can be used in standard assays known to those skilled in the art. In general, antibody is contacted with a sample under conditions which allow the antibody to bind ps20. Quantitation is conducted by conventional techniques known to those skilled in the art. These include, but are not limited to histochemical techniques EMIT, ELISA, latex agglutination immunoassays, FPIA and other immunoassay techniques. Useful antibodies to ps20 include polyclonal or monoclonal antibodies.
[0059] The following examples serve to illustrate specific embodiments of the invention, but should not be considered as a limitation on the scope of the invention.
EXAMPLE 1 Cloning and Sequencing of ps20[0060] This example describes the cloning and nucleotide sequence of rat ps20. To clone ps20 cDNA, a cDNA library was prepared from the rat prostate smooth muscle PS-1 cell line, which was a high expresser of ps20 as determined by Western analysis. The PS-1 cell line is described in U.S. application, Ser. No. 07/928,867 (now U.S. Pat. No. 5,496,800). The cDNA library was constructed in lambda ZAP Express vector from oligo d(T) primed cDNA. The ZAP Express vector was chosen based on versatility, ease of excision and recircularization to produce pBK-CMV phagemid subclones. The library exhibited anticipated representation of B-actin cDNA (as control) and IgG lectin-binding protein cDNA in test screening.
[0061] As an initial approach, degenerate primers were prepared based on the 5′ ends of the amino terminal sequence as determined from purified ps20 protein. Due to degeneracy of PCR probes, a nested PCR approach amplifying from 5′ vector primer to a ps20 degenerate primer followed by a nested amplification using ps20 forward and reverse degenerate primers allowed for amplification of authentic ps20 sequence (amino terminal end). The final PCR product had a predicted size of 81 bp, was cloned directly into pCR II plasmid by TA cloning. clones isolated, and clone 3438pCRII sequenced. The corresponding sequence was confirmed as ps20 by a direct match of the deduced amino acid sequence with the amino terminal sequence determined from purified ps20. Non-degenerate forward and reverse PCR primers were developed based on clone 3438pCRII sequence, and used in nested PCR reactions to amplify and clone the 3′ and 5′ ends of ps20 cDNA. The 5′ clone (clone T340pCRII) was 184 bp, and overlapped (by 50 bp) with clone 3438pCRII sequence plus an additional 120 bp of 5′ sequence. The 3′ clone (clone 42T7pCRII) was 868 bp in length, contained a 3′ poly A tail, and overlapped (by 54 pb) with clone 3438pCRII sequence.
[0062] Screening of PS-1 cDNA library was based on PCR to score positive plates followed by plaque hybridization with clone 42T7pCRII labeled insert (862 bp) as a probe to score individual colonies. A total of 1.2 million clones were screened with one positive clone detected in every 1,020,000 colonies. Clone 1025rps20pBK-CMV-1B was sequenced from both directions (sequence shown in FIG. 1), matched sequence from an additional separate clone 1025rps20pBK-CMV-2B, and confirmed as ps20 by deduced amino acid sequence identical to native ps20. Clone 1025rps20pBK-CMV-IB was 1029 bp in length and contained a 3′ poly (A) tail in agreement with Northern analysis of U4F cells and rat dorsolateral prostate which showed a single, identical sized species at approximately 1.1 kb as shown in FIG. 1. Sequence analysis indicated an open reading frame of 636 nucleotides beginning at nucleotide 52 (ATG), ending at nucleotide 688 (UGA stop codon), and coding for a deduced 212 amino acid protein. A hydrophobic leader sequence was predicted for amino acid 1-26 with a perfect signal peptidase cleavage site between Gly (#26) and Thr (#27) (−1 and +1 respectively) following the rules of von Heijne. (Von Heijne, G. 1984. J. Mol. Biol. 173:243-251). Thr (#27) (position +1 of mature secreted protein) through His (#54) were an exact match with Thr (#1) through His (#28) determined from the amino terminal of purified native ps20. Hydopathy analysis (Tmpred) suggested no transmembrane domain, predicting a secreted protein. No potential post-translational modifications were indicated with the exceptions of 5 potential casein kinase II sites. The cDNA clone predicts a mature, secreted protein of 20.7 kDa (identical to purified native 21 kDa ps20) and an intracellular molecular weight of approximately 23.6 kDa (including hydrophobic signal peptide) in close agreement with detection by Western analysis indicating an intracellular Mr of 29 kDa under these conditions. Subsequent refinements by the inventor suggest a Mr of 21-23 kDa under non-reducing conditions and a Mr of 27-29 kDa under reducing conditions. Larsen, et al. (1998) J. BIOL. CHEM. 273: 4574-4584.
[0063] Using clone 1025rps20pBK-CMV-2B as a labeled probe, a lambda gt11 I library prepared from normal human prostate gland (human prostate 5′-STRETCH cDNA, Clontech) was screened (1.2 million clones) with standard plaque hybridization techniques. Eight clones were isolated as potential full length, based on PCR screening of 5′ and 3′ ends. Of these, 5 were likely full length (1-1.2 kb) based on comparison to rat ps20 cDNA and the previous determination of human ps20 protein exhibiting an identical molecular weight to rat ps20.
[0064] The nucleotide sequence of rat ps20 is shown in FIG. 1 (SEQ ID NO. 5). Additionally, FIG. 7 depicts the nucleotide sequence of rat ps20 and which is referred to herein as SEQ ID No. 1. The sequence of FIG. 7 contains extra nucleotides in the 3′ uncoding region which is not depicted in the sequence shown in FIG. 1. Also shown in FIG. 1 is the deduced amino acid sequence of rat ps20 (SEQ ID NO. 6). The rat ps20 amino acid sequence is also shown in FIG. 8 and is referred to herein as EQ ID No. 2. The underlined portion represents a signal peptide (amino acids 1-26). The ps20 protein is encoded by a single 1.1 kb transcript expressed in U4F mesenchymal cell cultures and rat adult prostate tissue. The transcript codes for a 23.6 kDa protein having a predicted signal peptide leader sequence (aa 1-26) with a prototypical signal peptidase cleavage site prior to the first amino acid of the secreted purified protein.
[0065] Analysis of deduced amino acid sequence revealed that ps20 has a WAP-type four disulfide core domain, classifying ps20 as a novel member of the WAP-type four disulfide core domain protein family (ergo, the name of the gene family noted above). Cysteines 58-96 which participate in the WAP—type four disulfide core domain are also underlined in FIG. 1. The members of the WAP-type four disulfide core domain family are relatively small proteins containing a conserved 8 cysteine motif in the protein core involved in disulfide bonds. The majority of family members with known biological activity function as protease inhibitors. The family members having core domains most closely related to ps20 include: Chelonianin, 39.4% identity to ps20 (subtilisin protease inhibitor isolated from red sea turtle egg white); Antileukoproteinase 1, 35.4% identity to ps20 (HUSI-1, a secreted serine protease inhibitor); WAP, 35.3% identity to ps20 (whey acidic protein, a suspected protease inhibitor found in milk); WDNM1 protein, 33.3% identity to ps20 (a mammary gland metastasis-suppressor gene with predicted protease inhibitor function); HE4, 33.3% identity to ps20 (a predicted protease inhibitor secreted into epididymis); Kallman syndrome protein, 31.2% identity to ps20 (predicted protease inhibitor localized in extracellular matrix and required for proper olfactory and GnRH-synthesizing neuronal development); Elafin, 29.2% identity to ps20 (a secreted elastase-specific serine protease inhibitor); and Caltrin-like protein II, 27.1% identity to ps20 (a secreted protein from seminal vesicle inhibiting calcium transport into spermatozoa). FIG. 2 depicts the alignment of four disulfide core domain of ps20 with other family members. (1. SEQ ID NO. 7; 2. SEQ ID NO. 8; 3. SEQ ID NO. 9;4. SEQ ID NO. 10; 5. SEQ ID NO. 11; 6. SEQ ID NO. 12; 7. SEQ ID NO. 13; . SEQ ID NO. 14; 9. SEQ ID NO. 15.) Alignment scores were computed by a fasta scoring method. (EERIE).
[0066] Functional significance of this protein family points to roles in tissue modeling, cell differentiation and cancer metastasis control. WAP may play a role in terminal differentiation and development of mammary acinar epithelial cells. Directed expression of a WAP transgene by MMTV has resulted in impaired mammary gland development and a hyperplasia/dysplasia of the coagulating gland (anterior prostate gland) in male reproductive tract. This observation is of significance since it was observed that there was an increased staining intensity of ps20 in human BPH as compared to normal human prostate tissue.
[0067] Kallman syndrome produces an agenesis of olfactory bulbs referred to as “olfactogenital dysplasia” and a hypogonadotropic hypogonadism. The defective gene in Kallman syndrome is termed ADMLX and encodes a secreted protein containing the WAP-type four-disulfide core domain as well as fibronectin type III repeats. This protein may function in cell adhesion and as a protease inhibitor. ADMLX may participate in migration of GNRH neurons and the axonal extension of olfactory neurons, thereby inducing a differentiation pattern.
[0068] The WDNM1 gene is novel member of the four disulfide core protein family with proposed metastasis-suppressor functions. WDNM1 is down-regulated by 20-fold in rat metastatic mammary adenocarcinoma in comparison to non-metastatic mammary carcinomas. WDNM1 has been suggested to function as a protease inhibitor and hence, modulation of WDNM1 protein could result in unregulated protease activity, commonly associated with metastatic spread of carcinomas.
EXAMPLE 2 Preparation of Antibodies to ps20[0069] This example describes the preparation and characterization of antibodies specific to ps20. ps20 in rat and human tissues was localized with immunohistochemistry. A synthetic peptide was made based on positions 1-14 of ps20 peptide sequence and used as immunogen in female New Zealand rabbits following modifications of the procedures of Vitukiatus. Vitukiatis et al. J. Clin. Endocr. 33:988-991.
[0070] A 14 amino acid synthetic peptide corresponding to and unique to the amino terminus of purified ps20 was synthesized on an Applied Biosystems 430A Peptide Synthesizer: N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-C. For initial immunization, ps20 peptide was solubilized in sterile, tissue culture grade H2O (400 &mgr;g/ml) and mixed with Freund's complete adjuvant (1:1 ratio) and injected in 500 ml (100 &mgr;g) aliquots at multiple sites (4-5) in the neck (subcutaneous) and in the subscapular muscle tissue in the back (intramusclular) of three female New Zealand rabbits. At three weeks post primary immunization, sera samples were prepared and analyzed by solid phase enzyme linked immunoabsorbance assay (ELISA). Each antibody positive rabbit received a booster of 100 &mgr;g peptide in 500 &mgr;l of Freund's incomplete adjuvant injected subcutaneously into multiple sites of the back and neck, and an additional 100 &mgr;g intramusclular in the subscapular region. Serum samples were tested for ps20-specific antibody every 2 weeks and immunoglobulin subtype determined by ELISA analysis. Sera was tested for ps20 antibody at three weeks following the initial booster and antibody positive rabbits received a secondary booster following identical procedures. Sera from rabbits producing high titer antisera (activity at 1:106 dilution) was pooled and IgG was precipitated by ammonium sulfate (50% saturation), resolubilized in PBS, and dialyzed overnight against PBS at 4° C. The specificity of the antibody was confirmed by immunoreactivity with a 20 kDa protein in concentrated, partially purified preparations of conditioned medium from U4F cells, from which ps20 was purified.
[0071] Immunoreactive IgG was purified by peptide column chromatography. Peptide (10 mg) corresponding to the first 14 amino acids of purified ps20 was generated as described above, and coupled to 1 g CNBr-activated Sepharose 4B following standard procedures as described in Methods in Molecular Biology, Vol. 34, “Immunocytochemical Methods and Protocols”, Lorette C. Javois (ed.), Chapters 19-23, pgs.155-193, Humana Press, Totowa, N.J. 1994 and poured into a 2 ml Poly-Prep column (Bio Rad). IgG preparations were diluted in PBS buffer (200 mM sodium borate, 160 mM sodium chloride, pH 8.0) and chromatographed through the column two times sequentially. The column was washed extensively in PBS (10-15 column volumes) and bound antibodies eluted with glycine-Cl buffer (0.05 M glycine, 0.15 M NaCl, pH 2.28). Fractions (2 ml) were eluted and collected directly in tubes containing 0.5 ml neutralizing buffer (0.5M phosphate, pH 7.7). Fractions were assayed for protein content (absorbance at 280 nm) and peak fractions pooled and assayed for immunoreactivity by solid phase enzyme linked immunoabsorbance (ELISA) assay. Antibody production was scored by ELISA. High titer antisera was detected in 3 rabbits.
[0072] The inventor has also made similar anti-peptide antibodies to regions of the protein near the carboxyl terminus and middle regions (data not shown). Such anti-peptide antibodies exhibit characteristics (albeit, unique binding affinities) to that described in detail for the amino terminus anti-peptide antibody here. In all cases, these antibodies have the capacity to function in diagnostic assays and affinity purification methods against the protein target.
[0073] An IgG fraction of ps20 antisera was analyzed for specificity by Western analysis and affinity purified antibody prepared by gel chromatography using peptide 1-14 covalently attached to sepharose 4B. Western analysis indicated mono-specific reactivity with the ps20 (20-21 kDa) secreted form (minus signal peptide) in conditioned medium. See, Rowley, et al. 1995. J. Biol. Chem. 270:22058-22065. Western analysis from U4F cell extracts showed mono-specific reactivity with a 29 kDa protein (FIG. 3) representing the unprocessed cellular form (includes a 26 amino acid hydrophobic leader sequence) predicted to increase the apparent backbone (24.6 kDa) size in SDS-PAGE and Western analysis. (A) U4F fetal rat urogenital sinus mesenchymal cells. Lane 1: Coomasie stain, 2: ps20 antisera IgG fraction, 3: preimmune sera, 4: affinity purified ps20 antibody, 5: no primary antibody. The ps20 antisera IgG fraction (lane 2) specifically recognized a single species of apparent 29 kDa size under these conditions. The affinity purified antibody (lane 4) recognized the 29 kDa band exclusively. For refinements in these Mr; see above discussion. Preimmune sera (lane 3) recognized all non-specific cross-reactive bands. Secondary antibody alone showed no banding pattern (lane 5). Western analysis of adult rat prostate smooth muscle and human prostate smooth muscle extracts showed identical immunoreactivity to a 29 kDa band from both rat and human cell lines. These studies indicated rat and human ps20 forms were identical in size and immunoreactivity as predicted by nearly identical amino acid sequence.
EXAMPLE 3 Immunohistochemical Localization[0074] This example shows the immunohistochemical localization of ps20 in prostate specimens.
[0075] FIGS. 4E & F, 5, and 6 show ps20 localization in human prostate specimens. FIG. 4 shows ps20 immunohistochemical localization in rat prostate gland with affinity purified antibody. Immunohistochemical localization was conducted by the procedure described in Methods in Molecular Biology, Vol.34, “Immunocytochemical Methods and Protocols”, Lorette C. Javois (ed.), Chapters 19-23, pgs. 155-193, Humana Press, Totowa, N.J. 1994. Basically, two month old and six month old male Sprague Dawley rats were sacrificed and whole tissues fixed in formalin O.N. Fixed tissues were embedded in paraffin and cut into 5 &mgr;m thick sections that were applied to poly-L-lysine coated slides and baked at 37 C prior to staining.
[0076] Formalin-fixed, paraffin-embedded sections of human prostate gland from 31 patients were obtained from Methodist Hospital, Houston (18), and from Texas Children's Hospital, Houston (13). Sections of 5 &mgr;m thickness were cut from the paraffin embedded blocks and applied to poly-L-lysine coated slides. Slides from twenty four adult patients ages 53 to 72 generally characterized as: carcinoma (9), BPH (3), severe BPH (2), stromal BPH (3), and normal (6) were stained for ps20. Slides from four patients of less than one year of age and four patients with ages between 10-14 years were stained.
[0077] Tissue sections were deparaffinized by immersion in Hemo D 1×10 min. and 1×5 min.; rehydrated by 5 min. graded washes in 100%, 95%, and 70% ethanol; permeablized by immersion in 1×PBS/0.1% Triton-X-100 for 5 min.; and treated 5 min. with 3% peroxide (H2O2) (diluted from 30% Sigma) to minimize endogenous peroxidase activity. Primary antibody incubations were performed at the concentrations and conditions described for immunocytochemistry, with the exception that affinity purified ps20 antibody was incubated with tissue sections O.N. at 37° C. Immunoreactivity was visualized by a 45 min. incubation with either biotinylated goat anti-rabbit or goat anti-mouse secondary antibodies (Sigma), diluted 1:15; followed by a 30 min. incubation with ExtrAvidin-conjugated peroxdidase (Sigma), diluted 1:15; concluding with a 7 min. incubation with diaminobenzidine (DAB) and mounting with Gel Mount. Staining of slides with hemotoxalin and eosin (H & E) were performed as described in Methods in Molecular Biology, Vol. 34, “Immunocytochemical Methods and Protocols”, Lorette C. Javois (ed.), Chapters 19-23, pgs. 155-193, Humana Press, Totowa, N.J. 1994. Slides were analyzed by light microscopy (Labophot-2, Nikon) and photographed on Ectachrome 400 slide or Royal Gold 25 print film (Eastman Kodak).
[0078] In FIG. 4, Panel (A) depicts the affinity purified ps20 antibody, Panel (B) the negative control, Panel (C) the smooth muscle a-actin (SM &agr;actin), and Panel (D) hematoxylin and eosin staining patterns. ps20 antisera (A) and the ps20 IgG fraction showed the same specific immunolocalization in rat prostate periacinar smooth muscle. Negative controls, including no primary antibody (B), preimmune sera, and antibody preabsorbed with ps20 peptide, showed the same lack of specific staining. ps20 immunolocalized to a subset of SM a-actin positive cells. Immunolocalization is specific to smooth muscle, but is not specific to prostate. Strong staining was observed in the smooth muscle of other male reproductive tract tissues, including the vas deferens and seminal vesicle. Moderate staining was observed in the tunica media of arteries and the smooth muscle of colon and small intestine. No apparent staining was observed in the brain, lung, bladder, or testis. Immunolocalization of ps20 in human prostate Panel (E) showed a localization corresponding to a subset of SM a-actin positive cells as shown in Panel (F). Exclusive localization was observed in the periacinar smooth muscle cells immediately adjacent to epithelial acini. Significant reactivity was not observed in any other cell type. A survey of other tissues including seminal vesicle, vas deferens, stomach, intestine, lung, salivary gland, heart, brain and testis showed ps20 expression was preferential to smooth muscle. Highest reactivity was noted in male reproductive tract tissues (vas deferens, prostate, seminal vesicle) with moderate staining in smooth muscle of gut and tunica media of arteries. No reactivity was noted in testis, lung, or brain. Of interest, ps20 reactivity was observed in the tunica media of arteries in the prostate gland. Localization was specific to smooth muscle cells (a-actin positive stromal cells). The human, unlike rat, does not have a precise periacinar ring of smooth muscle cells around epithelial acini. Rather, human prostate stroma is a mix of smooth muscle and fibroblasts. The ps20 positive cells correlated with a-actin positive cells all sections. Of interest were the staining patterns observed in prostatic disease.
[0079] FIGS. 5 and 6 show ps20 localization in BPH and prostatic carcinoma consistently high relative to normal. In FIG. 5, Panel (A) depicts ps20 antisera IgG fraction, Panel (B) preimmune sera, Panel (C) SM a-actin, Panel (D) hematoxylin and eosin staining. Immunolocalization of ps20 in regions of BPH is similar to that in normal regions of the adult human prostate or is slightly elevated in comparison to normal. Strong to elevated staining was observed in patients having both glandular and stromal BPH. The staining pattern shown here is representative of sample evaluated (5 patients diagnosed with glandular BPH and 3 with stromal BPH.) In contrast, staining intensity of ps20 was very heterogeneous and generally lower in carcinoma samples. In FIG. 6, Panel (A) depicts ps20 antisera IgG fraction, Panel (B) preimmune sera, Panel (C) SM a-actin, Panel (D) hematoxylin and eosin staining. Immunolocalization of ps20 in regions of carcinoma exhibited a heterogeneous staining pattern relative to normal in nine carcinoma patients evaluated. ps20 staining was reduced in stroma surrounding some poorly differentiated nodules, as shown in Panel (B). In particular, ps20 staining intensity was low or absent altogether in some (not all) stromal regions adjacent to poorly differentiated carcinoma nodule located in the peripheral, subcapsular region as shown in FIG. 6.
EXAMPLE 4 Human ps20 Sequence[0080] This example describes the nucleotide and deduced amino acid sequence of human ps20. A commercially available (Clonetech, Palo Alto, Calif.) cDNA library was used. The library was prepared from normal human prostate gland. Screening was done using standard plaque hybridization procedures as described in Current Protocols in Molecular Biology, Vol. 1, Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D; Seidman, J. G.; Struhl, K.; and Smith, J. A. (eds.), John Wiley & Sons. NY, N.Y. 1995. Plaques were transferred to Nytran 0.45 &mgr;M membranes (Schleicher and Schuell, Keene, N.H.), DNA cross lined by the Stratalinker UV cross linker (Statagene), and membranes pre-hybridized 2 h in at 42° C. in the hybridization solution (50% formamide, 2×PIPES buffer, 0.5% (w/v) SDS, 100 &mgr;g/ml sonicated salmon sperm DNA). Clones 42T7pCRII insert was purified from an agarose gel slice by Spin-X columns (Corning Costar Corp., Cambridge, Mass.), 150 ng labeled with &agr;[32P]-dCTP (Amersham, Cleveland, Ohio) by random priming with Klenow Enzyme (labeling grade, Boehringer, Mannehiem, Indianapolis, Ind.). Unincorporated nucleotides were removed by NucTrap Probe Purification Column (Stratagene), and denatured probe hybridized with filters at 1-3×106 counts/ml overnight at 42° C. in hybridization cocktail. Filters were washed 3×10 min at room temperature in 2×SSC, 0.1% SDS, and 2×20 min at 55° C. in 0.2×SSC, 0.1% SDS and exposed to X-OMAT AR film (Eastman Kodak, Rochester, N.Y.). Positive plaques were screened by PCR, phagemids excised from lambda arms by the Rapid Excision Kit (Stratagene), and DNA prepared either by mini alkaline lysis or by Oiagen tip-500 (Oiagen, Chatsworth, Calif.). The probe was rat ps20 cDNA clones labeled with 32p Colonies of DNA were lifted via filters and hybridized to the rat ps20 cDNA clones. Positive colonies were selected and purified by second and third round screening.
[0081] Positive colonies were sequenced and compared to the rat sequence. Positive clones H1T2100 and H6B2-3 were sequenced using both dideoxy sequencing (as described in Current Protocols in Molecular Biology Vol 1, see above) and automated sequencing using the IBI model 377 automated sequenator. Clone H1T2100 was 1124 bp in size and contained nucleotide 1-1124 sequence which included the entire coding region (sequence encoding the mature ps20 protein). Clone H6B2-3 was approximately 1000 bp in size and contained the entire 3′ untranslated region (including the poly A tail) and the coding sequence (overlap with clone H1T2100) minus the first few amino acids. The human nucleotide sequence and derived amino acid sequence (the human cDNA sequence encoded a 220 amino acid protein) were compared to the rat ps20 sequences using the MacVector 4.1 sequence analysis program. Based on these analyses, the human and rat amino acid sequences (Positions 1-212, rat) were well conserved in sequence with a 82.1% direct match and a 90.6% overall similarity when considering conservative substitutions of amino acids. The human ps20 protein contains an extra seven amino acids in the amino terminal leader peptide sequence and an added amino acid at position #52.
[0082] The nucleotide sequence of human ps20 is shown as SEQ ID No. 3 in FIG. 9. The derived amino acid sequence of human ps20 is shown as SEQ ID No. 4 in FIG. 10.
EXAMPLE 5 Chromosomal Localization of ps20 Using Human and Mouse Gene[0083] The purpose of the present example was to identify the chromosomal location of the human gene encoding ps20 and to determine whether it maps to a site implicated in cancer or disease. The inventor first cloned the human ps20 cDNA from a prostate cDNA library and used it to isolate the human gene from a P1 human genomic library (as described above). By fluorescent in situ hybridization (FISH), the inventor mapped the human gene (WFDC1) to chromosome 16q24.3, a region of LOH in several human cancers, including prostate and breast. To facilitate future studies of ps20 function in vivo in mouse model systems, the inventor also cloned the mouse ps20 gene (Wfdc1) and sequenced it to determine its genomic structure. The mouse and human ps20 genes both consist of seven exons, suggesting the gene encoding ps20 is most similar to the largest gene in the WAP Signature domain family, the KAL gene, which is genetically linked to Kallman Syndrome. The amino acid sequences of mouse, rat, and human ps20 protein are highly conserved. Due to its location on chromosome 16q24.3, combined with its growth inhibitory properties, the inventor has identified ps20 as a novel candidate tumor suppressor gene.
[0084] Screening of Human ps20 cDNA. The Human Prostate 5′-STRETCH Lambda gt11 cDNA Library (Clontech, Palo Alto, Calif.) was screened by moderate stringency hybridization with a partial rat ps20 cDNA clone as probe. Ten 150 mm plates with approximately 45,000 pfu/plate were prepared, transferred to Nytran 0.45 mm membranes (Schleicher and Schuell, Keene, N.H.), and DNA cross linked by UV irradiation (Stratalinker, Stratagene, La Jolla, Calif.). Membranes were pre-hybridized 2 h at 42 C in hybridization solution (50% formamide, 2×PIPES buffer, 0.5% (w/v) SDS, 100 &mgr;g/ml sonicated salmon sperm DNA). The insert from clone 42T7-1pCRII (Larsen et al. 1998. J Biol Chem. 273:4574-4584.), corresponding to the 864 nt of C-terminal ps20 sequence, was excised and purified, and 150 ng a-[32P]dCTP (Amersham, Cleveland, Ohio) labeled by random priming with random hexamer primers and Klenow Polymerase (Boehringer Mannheim, Indianapolis, Ind.). The labeled probe was incubated with membranes at >1×106 cpm/ml in hybridization cocktail ON at 42 C undergoing constant rotation. Filters were washed 2×20 min at RT in 2×SSC, 0.1% SDS and 2×20 min at 55 C in 0.2×SSC, 0.1% SDS and exposed to X-OMAT AR film (Eastman Kodak, Rochester, N.Y.). Eight positive colonies were obtained and DNA prepared for each (Lambda DNA Maxi Prep Kit, Qiagen, Chatsworth, Calif.). Inserts were excised from the lambda arms by EcoRI restriction digestion and subcloned into the vector pBKCMV (Stratagene, LaJolla, Calif.). Five clones were confirmed to be partial human ps20 clones by sequencing and comparison with rps20 cDNA sequence.
[0085] Cloning of human ps20 genomic clone. Insert from human ps20 cDNA clone H1-1pBK-CMV was purified and used to screen a human P1 library (Ad10SacBII) by two sequential rounds of plaque hybridization (Genome Systems, St. Louis, Mo.). The putative clone was transferred into host NS1356 and DNA prepared by a modified alkaline lysis method (100 KB column method), as recommended by the manufacturer (Genome Systems, St. Louis, Mo.). The human ps20 genomic clone, H1pAd10SacBII (75-100 kB), was confirmed to contain the human ps20 gene by Southern blotting with a H1-1 cDNA probe and by sequencing with primers corresponding to human cDNA sequence (Genosys, The Woodlands, Tex.). Sequencing indicated the human genomic clone contains seven exons.
[0086] Fluorescence in Situ Hybridization. Chromosome spreads were prepared using phytohemagglutinin stimulated human peripheral blood lymphocytes, which were synchronized with methotrexate. Bromodeoxyuridine incorporation was used to optimize banding. Cells were harvested by standard cytogenetic techniques. Nick-translation of probes, in situ hybridization (Johnson et al. 1991. Am J Med Genet. 39:97-101.)(Lawrence et al. 1998. Cancer Epidemiol Biomarkers Prev. 7:29-35.), and gene localization analysis (Wydner et al. 1996 Genomics. 32:474-478.) were performed as described in detail previously. Metaphase preparations were hybridized with the human ps20 genomic P1 clone, H1pAd10SacBII, labeled with digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, Ind.). In some experiments the spreads were simultaneously hybridized with a second probe for telomeres labeled with biotin. Signals were detected by immunofluorecence with rhodamine-anti-digoxigenin and fluorescein isothiocyanate-conjugated avidin (Boehringer Mannheim) on DAPI (4,6-diamino-2-phenyl-indole)-banded chromosomes. Preparations were photographed with a Zeiss Axioplan fluorescence microscope equipped with a Photometrics CCD camera, filter wheel, and dual and triple bandpass filters (Chroma, San Juan Capistrano, Calif.), which avoids any optical shift between filters. Red and green hybridization signals were visualized on blue DAPI-banded chromosomes. Cytogenetic analysis was performed on images of contrast-reversed chromosomes to convert DAPI-bright bands to black and white G-dark bands, and signals as bars relative to the chromosome, with the bars approximating the precision of signal placement, as previously described (Wydner et al. 1996. Genomics. 32:474-478.)
[0087] Cloning of the mouse PS20 gene. Mouse genomic clones containing ps20 sequence were isolated by plaque hybridization from the mouse 129 SV Lamda Fix II genomic library with partial rat ps20 cDNA sequence. The mouse 129 SV Lamda Fix II library was constructed by cloning mouse genomic DNA partially digested with Sau3A into the Sau3A site in the Lambda Fix II vector. Initially, the library was screened with [32P]dCTP—labeled 42T7 (partial rps20 3′ end sequence) and one clone (MGLclone 2) was obtained. To obtain genomic clones containing 5′ ps20 sequence, the partial rps20 5′ end clone, T340 was used to identify two other mouse ps20 genomic clones, MGLclone 1 and MGLclone 3. Inserts were excised from the lambda library by digestion with XhoI and cloned into a modified pCR2.1 vector (lacking sequences including the 5′ EcoRI site through the BstXI site) at the XhoI site. Clones were screened for the presence of ps20 sequences by Southern analysis with the 42T7 and T340 probes. Complete sequencing demonstrated MGLclone 1 (SEQ ID No. 17) contains promoter sequence, exon 1, and part of intron 1. MGLClone 3 was partially sequenced and confirmed to contain exon 1 sequence and intron 1 (˜10 kb). Complete sequencing of MGLClone 2 (SEQ ID No. 16) indicated it contained part of intron 1 exons 2 through 7. Primers corresponding to the 3′ end of MGLClone 3 and the 5′ end of MGLClone 2 were used to amplify a fragment from 129 mouse genomic DNA by PCR under standard conditions. Sequencing of the fragment confirmed that MGLClone 3 and 2 are contiguous and separated by a Sau3A site.
[0088] Sequencing and analysis. Multiple pass DNA sequencing was performed using an Applied Biosystems Model 377 Sequencer Version 2.1.1 using AmpliTaq polymerase (Perkin Elmer, Branchburg, N.J.), 3 pmol primer (pBKT3-1, pBKT7-1, and ps20 specific primers), and 1 mg double-stranded DNA per reaction. Sequencing of the human genomic clone was accomplished using two-fold more primer and five-fold more DNA than for standard plasmid DNA sequencing Sequences were assembled using MacVector, 4.1 and AssemblyLign, 1.0 (Eastman Kodak, Rochester, N.Y.).
[0089] PCR primer sequences were designed using MacVector, 4.1 software. Nucleotide sequence searches were performed on all available databases using the BLASTN and TBLASTN (blast enhanced alignment utility) algorithms (Altschul et al. 1990 Journal of Molecular Biology. 215:403-410.) and modifications thereof. Secretory peptide prediction was confirmed by PSORT II (WWW Version). Analysis of ORFs was determined by WebGene and multiple sequence alignments performed with ALIGN (EERIE). Secondary structure predictions of deduced amino acid sequence were made by SOPMA (Self Optimized Prediction Method from Alignment) (Geourjon and Deleage, 1993. CABIOS. 9:87-91.) and SBASE (Pongor et al. 1994. Nucleic Acids Research. 22:3610-3615) and confirmed by BLASTX and TBLASTX searches of the available databases. Analysis of splice sites was performed using Splice View.
[0090] Cloning of Human ps20 cDNA.
[0091] The inventors screened a human prostate cDNA library with rps20 cDNA sequence as a probe in order isolate the cDNA encoding the ps20 homolog. Eight putative clones were obtained and sequenced, of which five were confirmed to be partial human ps20 cDNA clones by comparison with rat ps20 cDNA sequence. The three non-ps20 clones were very low homology DNA sequences, not closely similar enough to be related family members (data not shown). The two longest human ps20 cDNA cloncs, H1-1 and H1-6pBKCMV, were sequenced and compiled to generate the human ps20 cDNA 1366 nucleotide (nt) composite full length sequence (SEQ ID No. 3). Clone H1-1 corresponds to nt 1-1124 and H1-6 to nt 205-1366 of full length human ps20 sequence. Comparison of the human and rat ps20 cDNAs indicated the sequences are 58.1% identical. The most significant differences in the rat and human ps20 cDNA sequences are that the human 5′ untranslated region (UTR) is 104 nt longer and the 3′ UTR is 211 nt longer than the rat. Comparison of the human ps20 DNA sequence with existing databases revealed significant homology of human ps20 cDNA with a 436 nt human EST (GenBank H52970) cloned from a human fetal male 20 weeks post-conception liver and spleen cDNA library.
[0092] Human ps20 is highly homologous to rat ps20 on the protein level. FIG. 11 shows the human ps20 cDNA sequence with the predicted protein sequence. The 1366 nt human ps20 cDNA sequence contains a start codon at nucleotide (nt) 155, a stop codon at nt 817 (asterisk), and a polyadenylation signal (underline). The open reading frame predicts expression of a 220 amino acid protein with a predicted signal peptide cleavage site immediately following Ala 31 (arrow). The deduced protein sequence is numbered beginning with the first amino acid of the signal peptide. The Cys residues comprising the WAP signature domain are noted (grey highlight). The open reading frame (ORF) presented initiates at the second ATG. The first ATG in the human ps20 cDNA sequence is at nt 18, but the context is poor with a C in the −3 and an A in the +4 position and the predicted ORF is short and not homologous to rat ps20 protein. The second ATG was presumed to be the actual translation start site. The second ATG (nt 155) through the stop codon at nt 817, predicts a peptide of 220 amino acids in length 82.1% identical and 90.6% similar to the rat protein. The upstream ATG in the human ps20 cDNA appears unique to the human sequence; whether it is functional or not is not known. Upstream ATGs are sometimes involved in translational control mechanisms. Few vertebrate mRNAs have upstream ATGs (5-10%), although those that do are often involved in growth control. Interestingly, another WAP signature domain family member, Elafin, also has two ATGs, of which only the second appears to be functional. Like rat ps20, human ps20 contains a WAP Signature domain, which may be critical for its function.
[0093] Genomic Cloning of Human ps20 and FISH Mapping to Human Chromosome 16q24.3
[0094] Given the potential role of ps20 in growth control, it was of particular interest to identify its chromosomal location and to assess whether it corresponded to sites implicated in cancer or disease. The inventors isolated the human PS20 gene from a human P1 library by plaque hybridization with the human cDNA as probe. That the single clone obtained, of insert size 75-100 kb, contained ps20 sequence was confirmed by Southern analysis and by direct sequencing with human ps20 specific cDNA primers (data not shown). Using fluorescence in situ hybridization and the digoxigenin-labled H1pAd10SacBII P1 clone, normal metaphase chromosomes were probed to determine the gene's location. Chromosomal location was determined from analysis of 20 digital images of metaphase spreads that typically exhibited signal on both sister chromatids of two homologous chromosomes. The chromosome was identified as chromosome 16, with no evidence of additional sites elsewhere in the genome (FIG. 12A). The analysis of ps20 location on banded chromosomes clearly indicated the gene resides in band 16q24 (FIG. 12B). This site was of significant interest because of its frequent loss in certain cancers, with band 16q24.3 strongly implicated in prostate and breast cancers. Close scrutiny of over 20 chromosomes showed that ps20 signal most frequently localized to the distal portion of 16q24, encompassing minor bands 16q24.2 and 16q24.3. While there was some technical variation in the placement of the signal, for a number of chromosomes the signals were so telomeric that they actually appeared to be off the end of the chromosome, as reflected in the ideogram in FIG. 2E. Because of the importance of potential of the precise sub-band localization, 16q24.3, the inventor co-hybridized telomeric and ps20 probes to assess further the proximity of the gene to the very end of the chromosome. As shown in FIG. 12D, on metaphase chromosomes the signals were generally co-localized and not separated along the length and rarely across the width of the chromosome. In interphase cells where DNA is more distended and provides higher resolution, separate but very closely-spaced signals were resolved (e.g. nucleus in FIG. 12C). The presence of multiple telomere signals per nucleus precluded precise determination of average interphase distance. However, based on earlier work correlating known DNA distances to interphase and metaphase distance, even a conservative estimate of the interphase separation between ps20 and the telomere observed here indicates the ps20 gene is likely less than 2 Mb from the telomere. These results support localization of the ps20 gene in the most telomeric sub-band, 16q24.3.
[0095] Genomic cloning and structure of mouse ps20 gene (Wfdc1). Mouse ps20 gene was isolated and the genomic structure determined by sequencing. The ps20 mouse genomic clone was isolated by screening a mouse genomic lambda library with rat ps20 cDNA sequence by plaque hybridization. Three clones were isolated and confirmed to contain ps20 sequence by Southern hybridization (data not shown). By sequencing, MGLClone 1 (SEQ ID No. 17) was found to contain ps20 exon 1 sequence, part of intron 1 sequence, and an additional 6.3 kb of upstream sequences. Partial sequencing of MGLClone 3 indicated it overlaps with MGLClone 1 sequence at its 5′ end and contains exon 1 sequence and intron 1 (estimated size 8-12 kb). Complete sequencing of MGLClone 2 (SEQ ID No. 16) revealed ps20 exons 2-7 and an additional 2 kb of downstream sequence. MGLClones 2 and 3 were determined to be contiguous following PCR amplification of intervening sequence between from mouse total genomic DNA and sequencing of the PCR product. MGLClone 2 and 3 were separated by a Sau3A site, which was the site used to construct the library. FIG. 13A summarizes the genomic organization of the mouse ps20 gene and the contributions of the three clones. Exons are labeled in Roman numerals with coding sequence in black and non-coding sequence in open boxes. Exons and introns are drawn to scale. The three contributing Lamda clones are shown with clones 1 and 2 drawn to scale. Shown in FIG. 13B is the mouse ps20 gene sequence with corresponding amino acids indicated under the exon sequences and the location and size of intron sequences indicated. 5.5 kb of Wfdc1 promoter sequence is omitted from the figure. Exons are labeled (Roman numerals) and amino acid sequence shown in single amino acid code below exon nucleotide sequence. Introns are not shown, but their locations and sized (of total intron sequence) noted. The WAP Signature domain is underlined. The nucleotide sequence is labeled with the transcription start site designated +1. The mouse genomic sequence has been submitted to GenBank in two parts: The complete PS20 gene spans approximately 26 kb. Sequencing of exon-intron borders of the human genomic clone, H1pAd10SacBII, confirmed that the human ps20 gene has the same genomic structure (data not shown).
[0096] Comparison of rat, mouse, and human cDNA and protein sequences. Shown in FIG. 14 is an alignment of human ps20 and rat ps20 amino acid sequences together with the putative mouse ps20 amino acid sequence deduced from the mouse genomic sequence. The mouse ps20 protein sequence, deduced from the genomic clone by comparison with rat cDNA sequence, is shown aligned with both rat and human ps20 amino acid sequences. The sequences are highly homologous. The signal sequence cleavage sites are indicated (arrow). The full length human ps20 protein is 82.1% identical and 90.6% similar to rat ps20 protein. Cleavage of hps20 following Ala 31, generates a mature protein 88.0% identical and 93.5% similar to the rat ps20. Amino acids representing conservative amino acid substitutions are indicated. The WAP signature domain is indicated (underline).Rat ps20 protein has a signal sequence that was demonstrated to be functional since mature rat ps20 protein was expressed and secreted by COS cells transfected with a rat cDNA expression construct. Both human and mouse ps20 proteins also have highly hydrophobic putative signal sequences with predicted cleavage sites indicated. The different species of ps20 are most dissimilar in the signal sequences. 1 TABLE 1 Percent identity among ps20 cDNAs and proteins cDNA full length protein mature protein rat and mouse 88.0% 93.4% 95.7% rat and human 58.0% 79.1% 86.1% human and mouse 58.0% 80.5% 87.7%
[0097] Table 1 shows the homologies among the rat, human, and mouse cDNAs and proteins. The rat and mouse proteins are highly homologous: the mature proteins (lacking signal peptide) are 95.7% identical. Human ps20 is not as homologous: human ps20 is 86.1% identical to the rat and 87.7% identical to the mouse mature protein. The putative functional motif, the WAP Signature domain, is contained within the region of highest homology among the three species FIG. 14 (underlined), suggesting the WAP domain may be critical for ps20 function. The only other apparent potential functional motif in ps20 protein is an additional cysteine-rich region downstream of the WAP motif (aa 144-180 of human ps20) consisting of four cysteines. This cysteine-rich region shares no homology with other proteins and is unknown significance, yet is highly conserved in all three species. In general, the amino acid sequence of ps20 is highly conserved in human and rodent species, particularly in the WAP Signature motif.
[0098] Relevance of cloned sequences to diagnostics and treatment. The cytogenetic localization of a gene can provide important clues as to a possible involvement in specific diseases, especially particular types of cancer. There are numerous sites throughout the genome that show recurrent breaks or LOH (loss of heterozygosity) in cancer, however such breaks or deletions are not necessarily observed consistently or at high frequency in patients with that cancer. However, the band 16q24 is deleted at a particularly high frequency in certain cancers, providing strong evidence that this site harbors one or more tumor suppressor genes (TSG). The human ps20 cDNA was cloned from a prostate library and the protein is expressed in human prostate. The inventor has mapped the PS20 gene to within 2 cM of the telomere, and therefore within the 16q24.3-qter region of LOH. Because there is some discrepancy in the boundaries of the region(s) of most frequent LOH in prostate cancer on chromosome 16q24, there may be more than one TSG within the region. PS20, located at 16q24.3-ter, maps to a previously reported region of LOH in prostate cancer and, owing to its localization and growth inhibitory properties, can be considered a candidate TSG.
EXAMPLE 6 Effects of ps20 on Stromal Cell Phenotype[0099] ps20 Induces Alterations in Cell Shape.
[0100] The protein ps20 affected COS cell growth when stably expressed by smooth muscle cells. Thus ps20 may have an autocrine function on cells which express it in vivo. To determine if prostate stromal cells, which express ps20, are affected by ps20 in their growth, the PS-1 adult rat prostate stromal cell line was treated with ps20-His. PS-1 cells were seeded in Bfs (serum containing) medium at low density with no treatment (FIG. 15a) or in the presence of ps20-His (FIG. 15c) or vehicle (FIG. 15b). PS-1 cells (passage 13) were grown at low density in Bfs media in the presence of vehicle or 10 &mgr;l/ml hps20-His, grown for 48 h, and fixed/stained with crystal violet (CV). In the presence of recombinant human ps20 protein (hps20-His), PS-1 adult rate prostate stromal cells demonstrated an altered morphology. Cells become more scattered and contained greater numbers of filopodia and long cellular extensions. Cells were fixed and stained with crystal violet to facilitate analysis of cell shape. The ps20-His treated cultures displayed more filllopodia and other cellular extensions, analogous to the ps20 treated COS cell cultures. In general, PS-1 cell cultures are more spindly than COS cells and do not pack as closely or in a cobblestone pattern, yet the ps20 treated cells did appear to be more spread out on the culture dish and to have more cellular extensions than the untreated cells. To quantitate the change in cell shape, the cells were photographed and images analyzed by NIH Image software. The average cell area of ps20 treated PS-1 cells is less than vehicle treated or untreated cells (data not shown). These studies suggest that ps20-His induces shape changes on prostate stromal cells similar, though not identical, to shape changes induced on carcinoma cell lines.
[0101] Growth effects of ps20 on the PS-1 cell line. To determine if ps20 is growth inhibitory to PS-1 cells, PS-1 cells were treated with ps20-His, vehicle control, TGF-&bgr;1, or FGF-2 for 24 hours, pulsed with [3H]-thymidine, and [3H]-thymidine incorporation recorded as an indirect measure of cell growth. As shown in FIG. 16, PS-1 cells were not growth inhibited under these conditions (data not shown). However, when PS-1 cells are seeded at lower plate densities, slight to moderate inhibition may occur (data not shown). TGF-&bgr;1 was not growth inhibitory. Additional experiments over a period of 72 hours confirmed that ps20 does not affect PS-1 cell growth over longer time periods (data not shown). Cell counting experiments confirmed that [3H]-thymidine incorporation approximated cell growth characteristics (data not shown). FGF-2 was mitogenic to PS-1 cell growth and on vascular cell lines. These data suggest the effects of ps20 on cell shape and cell growth involve independent, cell specific pathways.
[0102] ps20 Promotes Spheroid Formation.
[0103] 1. COS Cells
[0104] Stable transfectant COS cell lines were observed to “pile” differently than either mock transfectant lines or COS parent cell lines when cultures were allowed to become post-confluent. COS cells are not completely contact inhibited. Once COS cells reach confluence, they continue to grow and form mounds of piled cells (FIG. 16a). At high density, COS cells stably transfected with ps20 cDNA piled up differently than COS cells or mock transfected COS cells. Ps20 stable transfectant cell lines (COS rps20-3) formed spheroids at high density, while COS and mock transfectant cells formed “pilles.” The ps20 transfectant cells piled on each other to form three dimensional spheroids (FIG. 16c). The spheroids seemed to be anchored with long cellular stress cables. The mock transfectant lines formed piles resembling those made by the parent cell line (FIG. 16b). These data suggest ps20 alters COS cell-cell adhesion and/or cell-matrix adhesion.
[0105] 2. PS-1 Cells
[0106] Prostate stromal cells, as well as other smooth muscle cell lines, are known to form spheroids. To determine if PS-1 cells preferentially formed spheroids in the presence of ps20, PS-1 cells were seeded in the presence of ps20-His, vehicle, or were left untreated. The cultures treated with ps20-His formed spheroids (FIG. 16f), but mock treated (FIG. 16e), or untreated cells did not (FIG. 16d), although the cells did pile up. In independent experiments, spheroids did form in untreated cultures, but to a lesser extent than in ps20 treated cultures. PS-1 cells express endogenous ps20 and can form spheroids under ideal conditions. Therefore, spheroid formation in the absence of ps20 was not unexpected.
[0107] ps20 promotes migration. Requirements for hillcock formation have been studied in mesangial cells and have been shown to include: ability to migrate, alterations in matrix synthesis, alterations in cell-matrix contacts. The promotion of spheroids by ps20 suggests ps20 stimulates one or more of the component activities required for spheroid formation. To specifically determine if ps20 affects cell migration, COS cells, mock transfectant, and ps20 transfectant cells were assayed for migration ability in a migration/wounding assay (Clyman, et al. 1992. EXP CELL RES. 200:272-284; Jones, et al. 1996. PROC NATL ACAD SCI USA. 93:2482-2487). The assay was performed by seeding cells at high density on glass coverslips so that they would quickly form a monolayer and thereby minimize differences in cell growth rates. A scrape was made (in triplicate) on each cell monolayer. Coverslips were fixed and stained with crystal violet at timepoints throughout the course of the assay. Random regions near the center of the coverslip were photographed and analyzed by NIH Image. A cell density within the wounded area was determined as an indirect measure of the number of cells that had migrated into the wound site. Cell number was previously shown to correlate with cell number in this assay.
[0108] The ps20 transfectant cell lines were observed to fill in the wound site faster than mock transfected or the parent cell line over a 60 hr period, as shown in FIG. 17. ps20 stable transfectant COS cell lines were compared with COS (untransfected) and mock transfectanat COS cell lines in an in vitro migration/wounding assay. As shown, cells were seeded in a monolayer, wounded, fixed/stained, photographed, and cell density in the remaining area estimated by image analysis with NIH Image at multiple timepoints. rps20 stable transfectant cell lines invaded the wounded area faster than mock transfectant cell lines. Results are typical of n=3 experiments. Given the time period of the assay, cells would have time to undergo more than one replication, suggesting differences in proliferation rates could be a factor in this assay. Note, however, that the migration rate for ps20 transfectant lines is consistently higher than controls at all timepoints. Since ps20 transfected COS cells are inhibited in proliferation, the enhanced migration promoted by ps20 cannot be explained by an differences in proliferation rate. Additionally, since the average cell area of ps20 transfectant cell lines is less than for the mock or non-transfected cells, the actual number of ps20 transfectant cells is underestimated and, therefore, effects of ps20 on migration are greater than is reported by this method. Migration is optimal at an intermediate cell attachment strength, which has been demonstrated for human smooth muscle cells on fibronectin and collagen, indicating that ps20 alters attachment strength, either to increase it or decrease it. Another factor to consider with this migration assay, is the fact that the substrate is glass in the presence of serum. Others have reported that serum does act as a substrate and largely behaves like a vitronectin substrate due to the 10 fold higher levels (200-300 (g/ml vs 30 g/ml) of vitronectin than fibronectin in serum. Since cells can migrate on existing substrates or can synthesize their own substrate, ps20 could alter attachment strength to the extracellular matrix either by affecting extracellular matrix receptors (integrins) or by altering synthesis of extracellular matrix by the COS cells.
[0109] ps20 Inhibits Polymerized SM a-actin Filaments in PS-1 Cells.
[0110] The inventor previously demonstrated that the adult rat prostate stromal cell line PS-1 expresses smooth muscle markers, including SM &agr;-actin, h1-calponon, desmin, and androgen receptor, a marker of smooth muscle in the prostate in fully defined media lacking serum (FIG. 18). PS-1 cells grown in fully defined media lacking serum (MO). After 24 h, PS-1 cells express very little of the smooth muscle markers, SM &agr;-actin (FITC)(left) and calponin (Cy-3)(right). After 96 h In the presence of TGF-&bgr;1 (50 pM), PS-1 cells express smooth muscle markers, SM &agr;-actin and calponin. Note that in the absence of exogenously added TGF-&bgr;1, PS-1 cells express increasing levels of smooth muscle markers with time, though not to the extent as seen in the presence of TGF-&bgr;1.
[0111] At early passage, PS-1 cells express all of the above markers, immediately after attachment, but, as has been reported for most smooth muscle cell lines, the cells dedifferentiate with successive passaging. In the absence of exogenously added TGF-&bgr;1, PS-1 cells at intermediate passage (P. 20-35) myodifferentiate and express smooth muscle markers, but on a longer time scale and not to the same extent. However, PS-1 will spontaneously differentiate over time, or the process can be accelerated by TGF-&bgr;1, as has been reported with other prostate stromal cell lines.
[0112] Coordinate with the myodifferentiation of PS-1 cells, measured by visualization of SM &agr;-actin and calponin filaments, is a change in cell shape. To investigate whether ps20 induced shape changes are related to those induced by TGF-&bgr;1, PS-1 cells (p.20) were grown for 72 h in fully defined media lacking serum in the presence of 50 pM TGF-&bgr;1 (FIG. 19b), vehicle control (FIG. 19a), or ps20-His (FIG. 19c). PS-1 cells (p.20) were grown for 72 h in fully defined media (MO)+recombinant ps20 or TGF-&bgr;1 and labeled with anti(SM)-&agr;-actin (FITC) and propidium iodine (PI). 3.84% of untreated PS-1 cells express polymerized SM &agr;-actin filaments, while 56.5% of TGF-&bgr;1 treated, and 0.00% of hps20-His treated cultures express polymerized SM &agr;-actin filaments. In the presence of ps20, the expression of smooth muscle markers by PS-1 cells is inhibited. Data shown is representative of n>3 experiments.
[0113] Cells were fixed, double-labeled with a smooth muscle marker, anti-SM &agr;-actin antibody (FITC), and propidium iodide to visualize total cells. Cells treated with TGF-&bgr;1 were flat and polygonal shaped with abundant stress fibers labeling with SM &agr;-actin, while untreated or ps20 treated cells were rounder with virtually no SM &agr;-actin staining detectable. SM &agr;-actin positive cells were counted as a percentage of total cells, which is shown graphically in FIG. 19d: 3.84% of untreated PS-1 cells expressed polymerized SM &agr;-actin filaments, while 56.5% of TGF-&bgr;1 treated and 0.00% of hps20-His treated cultures expressed polymerized SM &agr;-actin filaments. The prevention of polymerized SM &agr;-actin filaments by ps20 is consistent either with reduction of levels of monomeric SM &agr;-actin available in the cell or inhibition of monomer polymerization into filaments.
[0114] ps20 Inhibits Monomeric SM &agr;-actin Levels.
[0115] To determine if ps20 affected levels of total SM &agr;-actin protein available in the cell, total levels of SM &agr;-actin monomeric protein were measured in cell extracts by immunoblot analysis with anti-SM &agr;-actin antibody. Cell extracts were made from PS-1 cells treated with ps20, controls, or TGF-&bgr;1 in fully defined media for 72 hours. In cell extracts, levels of monomeric SM &agr;-actin decreased with ps20 treatment (FIG. 20a). Quantitation of protein levels by densitometry is shown graphically in FIG. 20b. FIG. 20a shows a Western analysis of monomeric SM &agr;-actin in PS-1 cell extracts. PS-1 cells (p.20) were seeded and allowed to attach overnight in Bfs media, then allowed to grow for 72 h in fully defined media lacking serum in the presence of (lane 1) vehicle control, (lane 2) hps20-His (2 ng/ml), (lane 3) hps20-His (20 ng/ml), (lane 4) TGF-&bgr;1 (50 pM), or (lane 5) &bgr;-Gal-His negative control. Cell extracts were harvested in RIPA buffer and equal volumes of each extract resolved by 15% SDS-PAGE, followed by Western analysis with anti-SM &agr;-actin antibody. Duplicate samples were loaded on a duplicate gel and Coomassie stained to control for protein loading (data not shown). Levels of monomeric SM &agr;-actin were decreased in hps20-His, particularly at the highest concentration.
[0116] FIG. 20b shows image analysis of SM &agr;-actin Western blot. The Western blot in FIG. 20a and a duplicate Coommassie stained gel were scanned and analyzed with NIH Imaging software. The density of each band was calculated and values plotted relative to a strongly Coomassie staining band representative of protein loading. Numbers correspond to lanes in A.
[0117] These data suggest ps20 is not simply inhibiting polymerization of monomeric SM &agr;-actin into filaments. From these data alone, it cannot be determined if ps20 is affecting the transcription of message, translation of protein, or protein stability.
[0118] Although during phenotypic modulation of vascular smooth muscle cells (VSM) SM &agr;-actin filaments decrease, levels of monomeric SM &agr;-actin mRNA are not altered. To determine if levels of SM &agr;-actin are affected during the myodifferentiation of PS-1 cells, total RNA was made from PS-1 cells under conditions identical for those used to make protein extracts. SM &agr;-actin levels were analyzed by Northern analysis with a SM &agr;-actin specific probe. Levels of SM &agr;-actin remained constant at 72 hours (data not shown) under all conditions. Together, these data suggest ps20 affects SM &agr;-actin at the level of protein turn-over, although effects on polymerization into filaments cannot be ruled out.
[0119] ps20 inhibits TGF-&bgr;1 mediated induction of SM &agr;-actin filaments. TGF-&bgr;1 mediated effects can be modulated by other growth factors and cytokines. To determine if ps20 can inhibit TGF-&bgr;1 mediated differentiation, PS-1 cells were simultaneously treated with 5 pM (sub-maximal) TGF-&bgr;1 and hps20-His (20 (1/ml) (FIG. 21c), FGF-2 (10 mg/ml) (FIG. 21d), &bgr;-Gal-His negative control (FIG. 21b), or vehicle (FIG. 21a) for 72 h in serum-free media. In the presence of 5 pM (sub-maximal) TGF-&bgr;1, PS-1 cells were examined for expression of SM-&agr;-actin (G) in the presence of vehicle, &bgr;-Gal-His negative control, hps20-His or bFGF (10 mg/ml) at 72 h in serum-free media (MO). Representative cell morphologies of each are shown at left: cells are labeled with immunoflurescent antibodies to: SM-&agr;-actin (FITC-G) and b-actin (Cy3-R). SM-&agr;-actin positive cells were counted as a percentage of total cells and data shown in a histogram. The SM-&agr;-actin positive cells represented: 25% (vehicle treated cells), 34% (&bgr;-Gal negative control treated cells), 3.2% (hps20-His, 20 &mgr;l/ml), and 2.83% (FGF-2, or bFGF). ps20-His inhibited SM &agr;-actin filaments in the presence of 5 pM (sub-maximal) TGF-&bgr;1. As shown with the other smooth muscle cell lines, bFGF similarly inhibited TGF-&bgr;1 induced expression of polymerized SM &agr;-actin filaments and demonstrated a morphology similar to ps20 treatment. The mechanism of bFGF effects on SM &agr;-actin is not known.
[0120] SM &agr;-actin filaments were labeled by direct immunofluorecence. SM &agr;-actin (FITC-G) and b-actin (Cy3-R). SM &agr;-actin positive cells were counted as a percentage of total cells and data, shown in the histogram in FIG. 7e. SM &agr;-actin positive cells were counted as a percentage of total cells: vehicle 25% (FIG. 21e), (-Gal-His control 34%, hps20-His 3.2%, and FGF-2 2.83%. FGF-2 inhibited TGF-&bgr;1 mediated induction of polymerized SM &agr;-actin filaments, as previously reported with human prostate stromal cells. Similarly, ps20-His inhibited SM &agr;-actin filaments in the presence of 5 pM TGF-&bgr;1 and produced a morphology similar to bFGF that of bFGF.
[0121] In the presence of 5 pM (sub-maximal) TGF-&bgr;1, PS-1 cells were examined for expression of SM &agr;-actin in the presence of vehicle or increasing concentrations of &bgr;-Gal-His negative control, rps20His (rat), or hps20-His (human) at 72 h in serum-free media (Mo), as shown in the previous figure (FIG. 21). Both rps20-His and hps20-His induced a dose-dependent decrease in the percentage of SM &agr;-actin positive cells detected in the presence of serum-free media, demonstrating the effects of ps20 are dose-dependent and species independent (FIG. 22: In the presence of 5 pM (sub-maximal) TGF-&bgr;1, PS-1 cells were examined for expression of SM-&agr;-actin in the presence of vehicle or increasing concentrations of &bgr;-Gal-His negative control, rps20His (rat), or hps20-His (human) at 72 h in serum-free media (MO), as shown in FIG. 21. Both rps20-His and hps20-His induced a dose-dependent decrease in the percentage of SM-&agr;-actin positive cells detected in the presence of serum-free media, demonstrating the effects of ps20 are dose-dependent and species independent.)
[0122] ps20 mRNA expression is stimulated by TGF-&bgr;1. PS-1 cells, like rat prostate smooth muscle in vivo express ps20, yet factors affecting ps20 mRNA expression are unknown. To address the question of regulation of ps20 mRNA, PS-1 cells were grown under fully-defined media conditions for 24 hours in the presence of 10 nM DHT (dihydrotestosterone), 25 pM TGF&bgr;1, 10 nM DHT+25 pM TGF&bgr;1, or 50 pM TGF&bgr;1. Total RNA was isolated from cells grown under these conditions and ps20 mRNA expression analyzed by Northern analysis (FIG. 23). Middle panel: &agr;-32P-labeled rat ps20 cDNA probe was used in Northern analysis to probe total RNA isolated from Adult rat prostate smooth muscle (PS-1) cells grown under fully defined media conditions for 24 hours in the presence of 10 nM DHT, 25 pM TGF&bgr;1, 10 nM DHT+25 pM TGF&bgr;1, or 50 pM TGF&bgr;1. Under these conditions, ps20 mRNA expression was not stimulated by DHT, but was stimulated by TGF&bgr;1 in a concentration dependent manner. An additional enhancement of TGF&bgr;1 stimulation was observed in the presence of DHT. Upper panel: &agr;-32P-labeled rat ddp 17, an androgen regulated transcript, was used to probe an identical blot as a positive control for androgen regulation. Lower panel: RNA transferred to the blot was stained with methylene blue as a loading control.
[0123] Under these conditions, ps20 mRNA expression was not detectable (data not shown), without stimulation, nor was expression stimulated by DHT (FIG. 21, lane 2). But ps20 mRNA expression was stimulated by TGF&bgr;1 in a concentration dependent manner (FIG. 21, lanes 3 and 5). An additional enhancement of TGF&bgr;1 stimulation was observed in the presence of DHT (Upper panel). As a positive control for androgen stimulation, an androgen regulated transcript (ddp17), was used to probe an identical blot as a positive control for androgen regulation. The blot was stained with methylene blue as a loading control (lower panel).
[0124] Many other variations and modifications may be made in the methods herein described, by those having experience in this art, without departing from the concept of the present invention. Accordingly, it should be clearly understood that the compositions and methods described in the foregoing description are illustrative only, and not intended as a limitation on the scope of the invention.
Claims
1. An isolated DNA molecule encoding a urogenital sinus derived growth inhibitory factor.
2. The DNA molecule of claim 1 wherein said DNA is cDNA.
3. The DNA molecule of claim 1 wherein said DNA is genomic DNA.
4. The DNA molecule of claim 1 wherein said DNA is human DNA.
5. The DNA molecule of claim 4 wherein said DNA is genomic DNA.
6. The DNA molecule of claim 1 wherein said DNA is mouse DNA.
7. The mouse DNA of claim 6 wherein said DNA is mouse genomic DNA.
8. The mouse genomic DNA of claim 7 wherein said mouse genomic DNA comprises the sequence of SEQ ID NOS. 16 or 17.
9. The DNA molecule of claim 1 wherein said DNA is rat genomic DNA.
10. An isolated DNA molecule encoding a WAP four-disulfide core domain 1 gene.
11. The DNA molecule of claim 10, wherein said gene is WFDC1.
12. The DNA molecule of claim 10 wherein said gene is Wfdc1.
13. A recombinant urogenital sinus derived growth inhibitory factor.
14. The factor of claim 13 wherein said factor is a human protein.
15. The factor of claim 13 wherein said factor is a rat protein.
16. The factor of claim 13 wherein said factor is a mouse protein.
17. An antibody immunologically recognizing urogenital sinus derived growth inhibitory factor.
18. The antibody of claim 17 wherein said antibody is a polyclonal antibody.
19. The antibody of claim 17 where said antibody is a monoclonal antibody.
20. A method of detecting the presence of DNA or RNA encoding a urogenital sinus derived growth inhibitory factor in a sample comprising:
- labeling any of the DNA molecules of claims 1-12, or a fragment thereof;
- contacting said labeled DNA or fragment with a sample containing DNA in a manner conducive to DNA-DNA or DNA-RNA hybridization; and, determining if said hybridization takes place by detecting the presence of said labeled DNA or fragment.
21. The method of claim 20 wherein said labeling is radiolabeling.
22. The method of claim 20 wherein said labeling is fluorescent labeing.
23. A kit for detecting the presence of DNA or RNA encoding a urogenital sinus derived growth inhibitory factor comprising:
- labeled DNA as in any of claims 1-12; and
- hybridization reagents.
24. A method of detecting the presence of a urogenital sinus derived growth inhibitory factor in a sample comprising:
- labeling any of the antibodies of claims 17-19, or an immunologically reactive fragment thereof;
- contacting said labeled antibody or fragment with a sample containing protein in a manner conducive to antibody-antigen cross reactivity; and
- determining if said cross reactivity takes place by detecting the presence of said labeled antibody or fragment.
25. The method of claim 24 wherein the labeling is radiolabeling.
26. The method of claim 25 wherein the labeling is fluorescent labeling.
27. A method of detecting the presence or localization of DNA or RNA encoding a urogenital sinus derived growth inhibitory factor in a chromosome, comprising:
- labeling any of the DNA molecules of claims 1-12, or a fragment thereof;
- contacting said labelled DNA or fragment with a chromosome in a manner conducive to DNA-DNA or DNA-RNA hybridization; and
- determining the presence or localization of said chromosonal DNA encoding said factor on said chromosome.
28. The method of claim 27 wherein the labeling is radiolabeling.
29. The method of claim 27 wherein the labeling is fluorescent labeling.
30. The method of claim 27 wherein the labeling is biotin labeling.
31. A biologically functional vector comprising a DNA molecule encoding a urogenital sinus derived growth inhibitory factor.
32. The vector of claim 31 wherein said DNA molecule is a human DNA.
33. The vector of claim 31 wherein said DNA molecule is a rat DNA.
34. The vector of claim 31 wherein said DNA molecule is a mouse DNA.
35. A host cell containing any of the vectors of claims 31-34.
36. The cell of claim 39 wherein said cell is prokaryotic.
37. The cell of claim 39 wherein said cell is eukaryotic.
38. A method of making a recombinant urogenital sinus derived growth inhibitory factor comprising:
- culturing a cell as in any of claims 35-37 in a medium;
- harvesting said cell from said culture or harvesting said cell culture medium;
- extracting said factor from said cell or said medium.
39. A method of treating a stromal cell comprising:
- contacting said cell with a urogenital sinus derived growth inhibitory factor.
40. The method of claim 39 wherein said stromal cell is a vascular cells.
41. The method of claim 39 wherein said stromal cell is a tumor cells.
42. The method of claim 39 wherein said stromal cell is an organ smooth muscle cell.
43. The method of claim 39 wherein said stromal cell is a stroma cell involved in fibrosis, wound repair or reactive stroma.
44. A method of genetic therapy comprising:
- introducing into a patient a vector capable of transfecting said patient with a gene encoded in said vector;
- said gene encoding a urogenital sinus derived growth inhibitory factor.
45. The method of claim 44 wherein said transfection is permanent.
46. The method of claim 44 wherein said transfection is temporary.
47. The method of claim 44 wherein said therapy is vascular therapy.
48. The method of claim 44 wherein said therapy is cancer therapy.
49. An affinity chromatographic medium comprising an antibody against a recombinant urogenital sinus derived growth inhibitory factor.
Type: Application
Filed: Sep 23, 2002
Publication Date: Apr 24, 2003
Inventor: David R. Rowley (Houston, TX)
Application Number: 10252340
International Classification: C12P021/02; C12N005/06; C12Q001/68; G01N033/53; C07H021/04; C07K014/705;
